The gastrointestinal peptide hormone ghrelin stimulates appetite in rodents and humans via hypothalamic actions. We discovered expression of ghrelin in a previously uncharacterized group of neurons adjacent to the third ventricle between the dorsal, ventral, paraventricular, and arcuate hypothalamic nuclei. These neurons send efferents onto key hypothalamic circuits, including those producing neuropeptide Y (NPY), Agouti-related protein (AGRP), proopiomelanocortin (POMC) products, and corticotropin-releasing hormone (CRH). Within the hypothalamus, ghrelin bound mostly on presynaptic terminals of NPY neurons. Using electrophysiological recordings, we found that ghrelin stimulated the activity of arcuate NPY neurons and mimicked the effect of NPY in the paraventricular nucleus of the hypothalamus (PVH). We propose that at these sites, release of ghrelin may stimulate the release of orexigenic peptides and neurotransmitters, thus representing a novel regulatory circuit controlling energy homeostasis.

Energy stores are held relatively constant in many mammals. The circuitry necessary for maintaining energy homeostasis should (1) sense the amount of energy stored in adipose tissue, (2) sense and integrate the multiple opposing signals regarding nutritional state, and (3) provide output regulating energy intake and expenditure to maintain energy homeostasis. We demonstrate that individual neurons within the paraventricular nucleus of the hypothalamus (PVH) are capable of detection and integration of orexigenic (neuropeptide Y [NPY]) and anorexigenic (melanocortin) signals, that NPY and melanocortins are functional antagonists of each other within the PVH in the regulation of feeding behavior, and that melanocortin administration within the PVH regulates both feeding behavior and energy expenditure. These data provide a cellular basis for the adipostat within neurons in the PVH that appear to be jointly regulated by NPY- and melanocortin-responsive neurons.

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Homeostatic regulation of energy balance in rodents changes dramatically during the first 3 postnatal weeks. Neuropeptide Y (NPY) and melanocortin neurons in the arcuate nucleus, a primary energy homeostatic center in adults, do not fully innervate the paraventricular nucleus (PVN) until the third postnatal week. We have identified two classes of PVN neurons responsive to these neuropeptides, tonically firing neurosecretory (NS) and burst-firing preautonomic (PA) cells. In neonates, NPY could inhibit GABAergic inputs to nearly all NS and PA neurons, while melanocortin regulation was minimal. However, there was a dramatic, age-dependent decrease in NPY responses specifically in the PA neurons, and a 3-fold increase in melanocortin responses in NS cells. These age-dependent changes were accompanied by changes in spontaneous GABAergic currents onto these neurons. This primarily NPYergic regulation in the neonates likely promotes the positive energy balance necessary for growth, while the developmental switch correlates with maturation of homeostatic regulation of energy balance.

SUMMARY1. The whole-cell voltage clamp technique was used to record calcium currents in the somatic membrane of rat cultured dorsal root ganglion neurones.2. Neurones were enzymatically isolated from animals of three age groups (neonatal. 2-7 days; adult, 7 months; and old, 30 months) and maintained in primary culture 3-14 days.3. The neurones isolated from neonatal and old rats showed two distinct types of Ca2+ currents, a low-threshold transient current and a high-threshold sustained current, whereas neurones from old rats showed only a high-threshold calcium current.4. The density of the high-threshold calcium current was 28 4 + 6-3 pA/pF (mean+s.E.M., n = 54) in neonatal, 39 1+72 pA/pF (n = 62) in adult and 11 0+±46 pA/pF (n = 64) in old dorsal root ganglion neurones.5. We found no difference in elementary high-threshold Ca2+ current characteristies in neurones from different age groups. The single-channel conductance was (with 60 mm Ca2+ in the recording pipette) 16 0 + 2-7 pS (mean + S.E.M., n = 9) in neonatal, 162± P7 pS (n = 11) in adult and 164+ P2 pS (n = 12) in old neurones.6. Current-voltage relations and kinetics of high-threshold calcium currents showed no detectable age-dependent difference.7. The run-down of high-threshold calcium currents in dorsal root ganglion neurones from old rats was practically insensitive to intracellular administration of cyclic AMP and ATP. The same intervention caused a significant deceleration of Ca2+ current run-down in the majority of neonatal and in some adult cells.8. We suggest that the disappearance of the low-threshold calcium current and reduction of high-threshold calcium current with ageing is due to a depression of calcium channel expression during late ontogenesis. The decrease of sensitivity of high-threshold calcium channels to phosphorylation by cyclic AMP-dependent protein kinase in aged neurones could also be a reason for altered turnover between silent and functional pools of calcium channels, which may underlie the agedependent decline in the density of high-threshold calcium channels.

We have recently shown that NPY and alpha-melanocyte-stimulating hormone, which potently induce or inhibit feeding, respectively, have opposing modulatory actions on GABAergic synapses in the medial parvocellular region of the paraventricular hypothalamic nucleus (mpPVN). Because this action might underlie the effects of NPY on feeding, we have examined the pharmacology of NPY responses using electrophysiological recordings. Focal electrical stimulation within the PVN elicited a GABA(A) synaptic response in some mpPVN neurons, which was reversibly inhibited by NPY in a concentration-dependent manner (EC(50) = 28 nM). NPY did not alter the response to the GABA(A) agonist, muscimol. Agonist responses to NPY analogs were not consistent with a single NPY receptor subtype; the most subtype selective agonists were less effective than the more broadly selective ones. Antagonist blockade of individual receptor subtypes partly inhibited NPY action, while fully blocking effects of selective agonists. Combining Y1 and Y5 antagonists blocked actions of NPY entirely, but the Y2 antagonist also completely blocked actions of NPY in some neurons. NPY inhibits GABA(A) synaptic transmission onto mpPVN neurons, but this can be mediated by three different NPY receptors. Controversy regarding the receptor or receptor subtypes involved in NPY-mediated feeding may arise from the multiple NPY receptors present.

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